One of the fundamental questions in human genetics concerns the mechanism by which loss or change of a given disease-related gene product results in the pathophysiology of the disorder. Classic galactosemia (MIM# 230400) is a potentially lethal inborn error of metabolism that results from impairment of the human enzyme galactose-1-phosphate uridylyltransferase (GALT, E.C.2.7.7.10). Despite lifelong dietary restriction of galactose, which is the current standard of care, a majority of patients with classic galactosemia nonetheless experience learning disabilities, primary ovarian failure, and abnormal speech, growth, and/or motor function; the biochemical basis of this pathology remains unknown. Furthermore, although a mouse model of GALT-deficiency demonstrates metabolic abnormality, these animals have failed to recapitulate any of the phenotypic complications observed in patients. In contrast, Saccharomyces cerevisiae deleted for GALT demonstrate both metabolic and clear phenotypic abnormalities upon exposure to galactose. The long-term goal of this project is to define the underlying mechanism of pathophysiology in galactosemia. The short-term goal of the proposed work is to exploit the genetic and biochemical facility of yeast, and the biochemical facility of human fibroblasts in culture, to determine the mechanism(s) of galactose toxicity in these model systems. The Specific Aims of this proposal are: (1) to identify and characterize novel genes involved in the pathway(s) of galactose toxicity and resistance in GALT-deficient yeast, (2) to determine the roles of specific candidate genes and metabolites in galactose toxicity and resistance in GALT-deficient yeast, and (3) to determine the roles of specific candidate genes and metabolites in galactose toxicity in GALT-impaired human fibroblasts in culture. The results of these studies will not only test existing hypotheses and expand our basic knowledge of galactose metabolism in yeast and human fibroblasts, they will identify novel candidates and enable the formulation of novel hypotheses to direct future studies of galactose toxicity in more complex systems, including patients.